Environmentally Induced Transgenerational …...epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance

Environmentally Induced Transgenerational EpigeneticReprogramming of Primordial Germ Cells and theSubsequent Germ LineMichael K. Skinner1*, Carlos Guerrero-Bosagna M. Haque1, Eric Nilsson1, Ramji Bhandari1¤,

John R. McCarrey2

1 Center for Reproductive Biology, School of Biological Sciences, Washington State University, Pullman, Washington, United States of America, 2 Department of Biology,

University of Texas at San Antonio, San Antonio, Texas, United States of America

Abstract

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerationalinheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of alteredepigenetic information between generations in the absence of direct environmental exposures. The primary periods forepigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetalgermline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female(F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e.great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were alteredtransgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinctbetween germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in thetestis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptionalalterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that alteredtransgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin inducedepigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigeneticinheritance are provided.

Citation: Skinner MK, Haque CG-BM, Nilsson E, Bhandari R, McCarrey JR (2013) Environmentally Induced Transgenerational Epigenetic Reprogramming ofPrimordial Germ Cells and the Subsequent Germ Line. PLoS ONE 8(7): e66318. doi:10.1371/journal.pone.0066318

Editor: Austin John Cooney, Baylor College of Medicine, United States of America

Received March 14, 2013; Accepted May 3, 2013; Published July 15, 2013

Copyright: � 2013 Skinner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by a NIH NIEHS grant to MKS. The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

¤ Current address: School of Biological Sciences, University of Missouri, Columbia, Missouri, United States of America

Introduction

Environmentally induced epigenetic transgenerational inheri-

tance of disease and phenotypic variation involves the germline

transmission of altered epigenetic information in the absence of

direct exposure [1,2]. The critical window for exposure is during

the period of epigenetic reprogramming of the developing germ

line coincident with the onset of fetal gonadal sex determination

[1,2,3]. The primordial germ cells (PGCs) undergo an erasure of

DNA methylation during migration to the genital ridge and

colonization of the fetal gonads and then the germline genome

initiates remethylation of DNA at the onset of gonadal sex

determination in a sex specific manner [4,5]. Previous research

demonstrated that exposure of an F0 generation gestating female

to the agricultural fungicide vinclozolin during PGC development

in the developing fetuses promotes epigenetic transgenerational

inheritance of disease [1,3] and epigenetic alterations in the F3

generation descendants [1,6]. Subsequently, a number of different

environmental toxicants have been shown to promote exposure

specific alterations in the F3 generation sperm epigenome (DNA

methylation) [7]. These include dioxin [8,9], a plastic mixture

(bisphenol A (BPA) and phthalates) [10,11,12], the pesticide

methoxychlor [1], a pesticide and insecticide mixture (permethrin

and DEET) [13], and a hydrocarbon mixture (JP8 jet fuel) [14]. In

addition to environmental toxicants, nutrition [15,16] and stress

[17,18] can promote epigenetic transgenerational phenotypes.

The primary site of action of these different environmental factors

must be in the germ line in order to promote epigenetic

transgenerational inheritance. This phenomenon has been dem-

onstrated in a wide variety of species including rats [1,3], humans

[19,20], mice [9,21], plants [22,23], worms [24,25], and flies

[26,27]. The current study used an outbred rat model [1] and the

agricultural fungicide vinclozolin [28] to promote the epigenetic

transgenerational inheritance of abnormalities that include testis

spermatogenic defects and male infertility [1,29], prostate disease

[3,30], kidney disease [3], behavior alterations (e.g. anxiety)

[18,31,32], mammary gland tumor development [3], immune

abnormalities [3], and ovarian disease [7,33].

The molecular mechanism starts with the induction of an

epigenetic alteration in the developing male germ line during fetal

gonadal sex determination that promotes a permanent alteration

in the germline epigenome (e.g. sperm) [1,2,6]. The germ line then

PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e66318

transmits this altered epigenome to the ensuing embryo, which

then leads to all tissues and cell types having altered transgenera-

tional transcriptomes and epigenomes that can be associated with

adult onset disease [2,34,35]. The altered germline epigenome

appears to be imprinted-like in that it escapes the normal erasure

of DNA methylation following fertilization to transmit the

epigenome transgenerationally in a parent-of-origin (male) specific

manner [2,36]. The current study was designed to investigate the

transgenerational effects on the F3 generation germ line to

determine if these cells maintain altered developmental program-

ming of the epigenome and transcriptome.

Germ cell development is initiated in mammals when primor-

dial germ cells (PGCs) are derived from the epiblast during

embryonic development and subsequently migrate to the devel-

oping genital ridges [37,38,39]. The PGCs then colonize the

indifferent gonads prior to gonadal sex determination shortly

before the initiation of differentiation into the male or female germ

line depending on the sex of the fetus [39]. After several mitotic

events in the developing ovary the female germ cells enter

prophase 1 of meiosis and form nests of primary oocytes that then

develop after birth (rodents) into primordial follicles [40]. In the

developing testis the germ cells continue to proliferate and

organize into the developing cords that will eventually develop

into seminiferous tubules at the onset of puberty [41]. As PGCs

enter the developing gonads DNA methylation is largely erased

and several days later global de novo methylation occurs to re-

establish the methylome in these cells. Certain regions of the

genome (e.g. imprinted genes) adopt sex-specific DNA methylation

patterns at this time [4,5]. In the fetal testis, the germ cells

continue to proliferate mitotically and then enter a mitotic arrest

near birth and resume proliferation a few days after birth in the

rodent [42,43]. At the onset of puberty the spermatogonia develop

at the basal surface of the seminiferous tubule and become the

stem cell for the spermatogenic process. The spermatogenic

process involves mitotic divisions leading to development of

spermatocytes that then enter meiosis to give rise to haploid

spermatids which differentiate into spermatozoa that are released

into the lumen of the tubule [44]. The final stage of maturation

occurs in the epididymis when the sperm develop the capacity for

motility [45]. Epigenetic alterations in DNA methylation have

been described during the spermatogenic and maturation

processes to facilitate subsequent developmental events.

The supporting somatic cell in the fetal gonad is the precursor

Sertoli cell that in the adult forms the seminiferous tubule and

provides physical support for the male germ cells [46]. The

interstitial cells include the Leydig cells in both the fetal and adult

testis. Factors that promote the epigenetic transgenerational

inheritance of disease prior to and during fetal gonadal sex

determination can involve direct actions on the PGCs or

subsequent germ cells, as well as indirect actions on the somatic

cells such as precursor Sertoli cells and Leydig cells that

subsequently influence the germ cells. Although the direct versus

indirect actions of the environmental factors in vivo cannot be

distinguished, the ultimate target cell required to facilitate

transgenerational inheritance must be the germ cells.

The experimental design used isolated PGCs from testes at

embryonic day 13 (E13) that correspond to the initiation of testis

development and a stage at which DNA methylation has been

erased from the germ cell genome. In addition, type T1

prospermatogonia were isolated from E16 testes at a stage

following cord formation when remethylation of the germline

genome has begun [47]. The current study examined the

transcriptome and epigenome (DNA methylation) in germ cells

from these two stages of development in F3 generation control and

vinclozolin lineage transgenerational animals. The objective was to

obtain insights into germline epigenetic programming and its role

in the epigenetic transgenerational inheritance phenomenon.

Results

The experimental design involved the exposure of an F0

generation gestating female outbred Spague-Dawley rat during the

period of embryonic days 8–14 that correlate with the end of

primordial germ cell (PGC) migration and early events of gonadal

sex determination [1,2]. The F0 generation females were exposed

to a vehicle (dimethylsulfoxide DMSO) as control or to

vinclozolin, as described in the Methods. The F1 generation

offspring were bred to generate the F2 generation and the F2

generation offspring were bred to generate the F3 generation

offspring, as previously described [3]. No sibling or cousin crosses

were used to avoid any inbreeding artifacts. The timed pregnant

F2 generation females were used to isolate the F3 generation

control and vinclozolin lineage fetal gonads at the E13 and E16

time points. The F3 generation E13 PGC and E16 prosper-

matogonia were isolated as described in the Methods and found to

be $85% pure based on morphological criteria [48]. RNA and

DNA were isolated from the freshly isolated cells to examine gene

expression by microarray analysis and DNA methylation by

methylated DNA immunoprecipitation (MeDIP) followed by

analysis on a genome-wide promoter tiling array (Chip) using a

comparative hybridization MeDIP-Chip analysis between control

and vinclozolin lineage samples [6] as described in Methods. This

allowed a comparison of the transcriptome or epigenome

alterations in F3 vinclozolin lineage germ cells at E13 and E16.

Three separate experiments involving different sets of animals and

different germ cell isolations were analyzed with three different

microarray and MeDIP-Chip analyses.

The germ cell transcriptome analyses demonstrated that all

arrays were of good quality with no abnormal hybridization

detected, Figure S1. Differential gene expression between control

and vinclozolin lineage F3 generation germ cells was determined

as previously described [34]. There were 592 differentially

expressed genes in germ cells at E13 and 148 differentially

expressed genes in germ cells at E16, Figure 1A. The complete lists

of differentially expressed genes are presented in Tables S1 and

S2. Interestingly, comparison of the gene sets between E13 and

E16 identified only 25 genes common to both lists. Observations

demonstrate the majority of differentially expressed genes are

distinct between these two developmental stages, Figure 1A and

Tables S1 and S2. The differentially expressed genes identified at

each stage were clustered into functional categories as presented in

Figure 1. Observations demonstrated the E13 and E16 gene sets

generally had similar gene categories represented for both the up

and down regulated genes. Although the specific E13 and E16

germ cell differentially expressed gene sets are primarily distinct,

overlap was observed in major functional gene categories affected.

The specific gene category for each gene is presented in Tables S1

and S2.

The E13 and E16 germ cells differentially expressed gene sets

were analyzed for specific cellular pathways and processes as

previously described [34], see Methods. A list of cellular pathways

and processes that have three or more genes from the gene sets is

presented in Table 1. Interestingly, 24 different pathways were

identified for the E13 differential gene expression set, but only one

pathway had three or more genes for the E16 gene set. Therefore,

the genes in the E16 list were more widely distributed and not

enriched for specific pathways, while the E13 gene set did show

enriched participation in specific pathways, Table 1. A unique

Transgenerational Primordial Germline Epigenetics




affected pathway identified from the E13 differentially expressed

gene set was the olfactory transduction pathway, which had 64

different genes enriched in this pathway. As shown in Figure 2, all

of these genes were olfactory receptors that have been shown to

require critical epigenetic regulation [49,50].

A final analysis of the E13 and E16 differentially expressed

genes identified a gene network based on previous literature

involving binding and functional interactions between the various

genes in the specific gene sets, see Methods. A gene network for

the E13 gene set is shown in Figure 3A and identifies a number of

extracellular regulators, signaling molecules and transcription

factors that integrate functionally. In contrast, the E16 differential

expression list generated a small network with only six genes,

Figure 3B. Vinclozolin was found to induce altered transgenera-

tional germline transcriptomes that are distinct in PGCs and

prospermatogonia.

Genomic DNA from the E13 and E16 germ cells was isolated

and used to identify altered differential DNA methylation regions

(DMR) (epimutations) between the F3 generation control versus

vinclozolin lineage germ cells with MeDIP-Chip analyses. One

approach used in this type of analysis is to take the data from three

different experiments and generate an average mean to assess

statistical significance. This approach generated 257 DMR for the

E13 PGCs and 242 DMR for the E16 germ cells with a statistically

significant difference (p,1024), Figure 4A. These DMR are the

result of what is termed an ‘‘average’’ analysis. In contrast a

second approach, previously used by our laboratory, used a more

stringent analysis requiring a reproducible and statistically

significant (p,1024) epimutation (i.e. DMR) to be present in

each separate experiment. This is termed an ‘‘intersection’’

analysis. This intersection analysis identified 24 DMR for E13

PGCs and 13 DMR for E16 germ cells, Figure 4B and Table 2.

Interestingly, very few of the DMR overlapped between the E13

and E16 germ cell datasets. The intersection DMR had one

overlapped promoter gene, Figure 4, identified as Pigb, Table 2.

The average DMR datasets had seven overlapped gene promoter

in both E13 and E16 germ cells. These were identified as Pigb,

Hmx2, Trx3, LOC499585, H1f0, Pim3 and Nign3, Table 2.

Therefore, as observed with the differential gene expression

datasets, Figure 1, the differential DNA methylation regions

Figure 1. Genes with mRNA expression levels significantly different between control and vinclozolin lineage F3 generation germcells at E13 and E16. (A) Number of differentially expressed genes unique to E13 PGCs, unique to E16 prospermatogonia, and common to both. (Band C) Numbers of differentially expressed genes in germ cells categorized by function for; (B) E13 and (C) E16.doi:10.1371/journal.pone.0066318.g001

Figure 2. Olfactory Transduction Pathway showing olfactory receptor genes differentially expressed between F3 generation E13PGC vinclozolin and control lineage rats. Adapted from Kyoto Encyclopedia of Genes and Genomes pathway rno04740.doi:10.1371/journal.pone.0066318.g002



(DMR) also had negligible overlap between the E13 and E16 germ

cell samples. Interestingly, a comparison with mature sperm

epimutations previously identified [6] demonstrated no overlap

with the E13 or E16 germ cell epimutations. The chromosomal

locations of the intersection DMR for E13 and E16 germ cells are

presented in Figure 4C. The majority of the autosomes and the X

chromosome contained one or more DMR.

The intersection DMR for both E13 and E16 germ cells were

next investigated for potential genomic features associated with the

genomic regions of each DMR, as previously described [6,7].

Common DNA sequence motifs identified in 100% of the

intersection DMR are shown in Figure 5A for both forward and

reverse strands of DNA. These motifs are similar to an

environmentally induced differential DNA methylation region

motif 1 (EDM1) previously associated with vinclozolin induced

transgenerational sperm DMR in the F3 generation [6]. The A

rich motif is similar to those known to associate with high mobility

group (HMG) box proteins that bind and bend DNA [51].

Another genomic feature investigated involves the CpG density

(CpG/100 bp) within each DMR. Interestingly, all the intersec-

tion DMR had less than 10 CpG/100 bp, with the majority being

only 1–2 CpG/100 bp, Figure 5B. Previous studies have

demonstrated that environmentally induced DMR in F3 genera-

tion sperm have a low CpG density of ,15 CpG/100 bp [6,7].

Therefore, the transgenerational E13 and E16 germ cell DMR

have similar genomic features as sperm DMR of a sequence motif

EDM1 and low density CpG density.

Analysis of the locations of the germ cell DMR (Table 2) with

the differentially expressed genes (Tables S1 and S2) demonstrated

no obvious correlation in either the E13 or E16 germ cell data sets.

Therefore, the DMR found in specific gene promoters do not

appear to regulate the adjacent genes expression at these stages of

germ cell development. As previously observed (32), the presence

of a DMR (epimutation) in a promoter does not generally correlate

to altered expression of the adjacent gene. An alternate

consideration is that indirect interactions between the DMR and

gene expression may exist. The approach employed datasets from

previous literature describing gene binding and functional

relationships to identify potential indirect correlations between

the germ cell DMR and differentially regulated gene sets, Figure 6.

A number of DMR were found to be indirectly correlated with the

germ cell differentially expressed genes at both the E13 and E16

stages of development. Therefore, minimal direct regulation of

gene expression was observed in the DMR associated genes, but a

number of potential indirect interactions were identified.

Table 1. Pathways enriched with F3 generation E13 and E16 germ cell gene lists.

Pathways Enriched with F3 generation E13 PGC gene lists

Pathway Name # genes affected

Olfactory transduction 64

Autoimmune thyroid disease 6

Measles 6

Systemic lupus erythematosus 6

Transcriptional misregulation in cancer 6

Allograft rejection 5

Calcium signaling pathway 5

Intestinal immune network for IgA production 5

Rheumatoid arthritis 5

Staphylococcus aureus infection 5

Viral myocarditis 5

Asthma 4

Neuroactive ligand-receptor interaction 4

Amoebiasis 3

Cell adhesion molecules (CAMs) 3

Fc gamma R-mediated phagocytosis 3

Glycolysis/Gluconeogenesis 3

Graft-versus-host disease 3

HTLV-I infection 3

Influenza A 3

Natural killer cell mediated cytotoxicity 3

Regulation of actin cytoskeleton 3

Tuberculosis 3

Type I diabetes mellitus 3

Pathways Enriched with F3 generation E16 germline gene list

Glutamatergic synapse 3

doi:10.1371/journal.pone.0066318.t001



Figure 3. Gene network of known relationships among those genes differentially expressed in control- compared to vinclozolinlineage F3 generation germ cells. (A) E13 network, (B) E16 network. Gene node shape code: oval and circle – protein; diamond – ligand; irregularpolygon – phosphatase; circle/oval on tripod platform – transcription factor; ice cream cone – receptor. Red colored nodes are up-regulated genes,blue color are down-regulated genes. Grey connecters represent general regulation, blue – expression regulation, purple – binding, green – promoterbinding, orange – microRNA effect. Cell membrane, nucleus, mitochondria, endoplasmic reticulum and golgi localizations are indicated. Network wasderived using Pathway StudioTM software.doi:10.1371/journal.pone.0066318.g003



Discussion

Environmentally induced epigenetic transgenerational inheri-

tance requires germline transmission of altered epigenetic

programming between generations in the absence of direct

environmental exposures [2,52]. A variety of environmental

exposures, including toxicants [7] and nutrition [15,16], can

promote transgenerational phenotypes [2,36]. The initial obser-

vations with vinclozolin indicated the transgenerational disease

phenotype is transmitted through the male germline (sperm), but

not through the female germ line (egg) [1]. The majority of

subsequent studies have focused on paternal (sperm) transmission

[7,33]. Preliminary observations demonstrate DDT induces

transgenerational phenotype (e.g. obesity) through the female

germ line. Therefore, environmentally induced epigenetic trans-

generational inheritance can involve either the male and/or

female germ cells.

The critical window of exposure for induction of transgenera-

tional epimutations in the mammal is during the later stages of

primordial germ cell migration and colonization of the fetal gonad

and during the initial stages of gonadal sex determination [1,3,29].

The PGCs undergo an erasure of DNA methylation prior to

gonadal sex determination and then subsequent re-methylation in

a sex-specific manner [4,5,37,38,39]. The hypothesis tested is that

Figure 4. Number of vinclozolin induced transgenerational DMR detected in F3 generation E13 and E16 germ cells, using twodifferent bioinformatic analyses. (A) The analysis is performed by averaging data from three comparative MeDIP-Chip per developmental timeusing a statistical cut-off of p,1024. (B) The analysis is performed by selecting only the DMR that repeatedly appeared as significantly changed in allMeDIP-Chip comparisons (intersection) using a cut-off of p,1024. (C) A graphical representation of the DMR location in all chromosomes in the ratthat were obtained through the intersection analysis for both E13 and E16 germ cell DMR.doi:10.1371/journal.pone.0066318.g004



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the exposure alters this epigenetic programming of the germline to

develop new epimutations in the form of differential DNA

methylation regions (DMR) that then, in a permanent imprint-

ed-like manner, are transmitted to subsequent generations to

promote the epigenetic transgenerational inheritance of disease

and phenotypic variation [2,36]. In the rat, the E13 stage of

development involves the final stage of PGC development and

initiation of gonadal sex determination when male germ cells

transition to prospermatogonial cell differentiation. The exposure

period during E8–E14 predominantly impacts PGC development.

In contrast, at E16 the male germ cells are at the type T1

prospermatogonia stage which occurs following testis cord

formation (42). The current study was designed to examine the

transgenerational transcriptome and epigenome alterations at

these two stages of germ cell development. The abnormal germ

cell programming observed in F3 generation vinclozolin lineage

descendants provides evidence that the original exposure to

vinclozolin induces an altered germ cell epigenome that is

transmitted transgenerationally.

Observations demonstrated 592 differentially expressed genes in

vinclozolin lineage germ cells at E13, but only 148 differentially

expressed genes at E16. PGCs at E13 have entered a unique

‘‘epigenetic ground state’’ represented by maximum erasure of

DNA methylation [53,54], whereas type T1 prospermatogonia at

E16 are at a stage when global remethylation of the germline

genome has initiated [37]. Therefore, a greater degree of DNA

methylation erasure in germ cells at E13 relative to E16 may be

related to the greater number of dysregulated genes detected at

these stages of development. Interestingly, there was negligible

overlap between the sets of dysregulated genes at these two stages,

with only 25 genes in common, Figure 1 and Tables S1 and S2.

Therefore, the E13 and E16 germ cells had predominately distinct

transgenerational transcriptomes. Although there was negligible

overlap in the transgenerational transcriptomes between the E13

and E16 stages, the majority of the functional gene categories

impacted were similar, Figure 1. Therefore, the vinclozolin

induced transgenerational germline transcriptomes appear to

impact similar cellular processes in the E13 and E16 germ cells

even though the specific gene sets affected are distinct.

A pathway analysis demonstrated the E13 gene list had over 20

different pathways altered while the vinclozolin lineage germ cells

at E16 had only one pathway disrupted. The E13 germ cell

differentially expressed genes influenced a variety of cellular

pathways and processes which were found to be distinct from the

E16 germ cells. One pathway that had 64 genes differentially

expressed in vinclozolin lineage E13 germ cells was the olfactory

transduction pathway, Figure 2. All the dysregulated genes encode

olfactory receptors that have been shown to be under epigenetic

regulation of the gene family [49,50]. Olfactory receptors have

been shown to be susceptible to transgenerational alterations

[18,34], and have also been shown to be involved in germ cell

function and reproduction [55]. Future studies will need to

examine the potential functional impact of the altered olfactory

receptor family. Interestingly, a previous study demonstrated that

sexual selection mate preference behavior was altered in the

transgenerational F3 generation vinclozolin lineage animals [31],

and it was speculated that this could in part be due to altered

olfaction [31]. The current study supports the potential that

altered regulation of the olfactory receptor gene family may

contribute to the behavior modifications in vinclozolin lineage

animals observed.

The final investigation of the germ cells transcriptome involved

a gene network analysis. The E13 PGC and E16 prosper-

matogonia germ cell differentially expressed gene sets were used to

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create networks of genes based on interactions among genes and

proteins reported in previous literature. A relatively large gene

network was identified in differentially expressed E13 PGC genes,

while at E16 the genes disrupted in germ cells formed a much

smaller network. Future studies are now required to assess the

functional importance of these transgenerational differentially

expressed gene networks. Observations demonstrate a transge-

nerational effect on PGC and prospermatogonia germ cell

transcriptomes.

The transgenerational PGC and prospermatogonia epigenomes

were also assessed in F3 generation control versus vinclozolin

lineage E13 and E16 animals. The germ cells from each

developmental period showed predominantly unique, non-over-

lapping, differential DNA methylation regions (DMR). The

Figure 5. Genomic features of the DMR identified. (A) The forward and reverse sequence motifs obtained with the MEME suite tool GLAM2 forthe vinclozolin induced transgenerational DMR from E13 and E16 F3 generation germ cells. (B) Shows the distribution of CpG sites (CpG/100bp) invinclozolin induced transgenerational DMR obtained from both the E13 and E16 germ cells.doi:10.1371/journal.pone.0066318.g005



analysis of DMR based on averages of three different experiments

generated larger sets of DMR for each period, but still showed

minimal overlap between E13 and E16 germ cells, Figure 4. Those

DMR that were reproducible for all experiments were termed

intersection DMR. Intersection DMR were almost entirely unique

for the E13 and E16 germ cells with only one overlap which was

found in the Pigb gene promoter. The prodigiosin biosynthetic

gene cluster family member Pigb encodes phosphatidylinositol

glycan class B. This protein has a variety of functions in a number

of species [56,57] from conferring mercury and copper resistance

[58,59] to facilitating signaling in cells [60,61]. Therefore, the

epigenetic alterations observed in vinclozolin lineage E13 PGCs

were distinct from those in E16 prospermatogonia. Observations

suggest an ongoing cascade of epigenetic alterations as germ cells

develop during this period. Interestingly, the DMR identified in F3

generation vinclozolin lineage fetal germ cells showed similar

genomic features as those previously described for F3 generation

vinclozolin lineage sperm [6]. These include low CpG density and

the presence of an adenosine rich DNA sequence motif, Figure 5.

The number of CpG/100 bp was found to average 3.1 CpG/

100 bp, such that these regions are CpG deserts, as previously

described [6,7]. Since C to T conversions are the most common

base pair mutation in mammals, evolutionarily deserts of CpG

develop in the mammalian genome [62]. The persistence of

regions retaining clusters of CpGs suggests potential regulatory

roles for these sites. In addition to low CpG density, many of the

DMR shared a DNA sequence motif similar to the environmen-

tally induced DNA methylation sequence motif1 (EDM1) previ-

ously identified in F3 generation vinclozolin lineage sperm [6].

Therefore, there appear to be specific genomic features that

renders these sites susceptible to become transgenerationally

programmed.

The current study demonstrates that ancestral exposure of a

gestating female during fetal gonadal sex determination to the

agricultural fungicide vinclozolin promotes a transgenerational

alteration in germ cell epigenetic programming in the F3

generation (great grandchildren). The E13 PGC at the onset of

fetal gonadal sex determination and the E16 prospermatogonia

both showed transgenerational alterations in both their transcrip-

tomes and epigenomes (DNA methylation), but these alterations

were largely distinct between the two developmental stages.

Therefore, the altered germ cell programming appears to involve a

cascade of transcriptional and epigenetic events that promote

germ cell mediated transgenerational inheritance [2,35]. In

addition to the DMR being distinct in vinclozolin lineage germ

cells at E13 and E16, these DMR were both distinct from those

previously identified in F3 generation vinclozolin lineage sperm

[6]. Observations demonstrate a specific DMR is not programmed

in the PGC and then the same DMR transmitted to the sperm, but

instead a cascade of epigenetic and transcriptional events

throughout germ cell development and spermatogenesis likely

leads to the mature sperm DMR transmitting the epigenetic

transgenerational phenotype. The current study used an MeDIP-

Chip genome wide promoter analysis and did not investigate the

entire genome. Therefore, DMR outside of promoter regions may

show more similarity between the PGC and the sperm DMR, but

this remains to be investigated. Future studies will require genome

wide analysis to identify the cascade of epigenetic and transcrip-

tional events at various germ cell developmental stages to correlate

with the DMR in the mature germ cells.

Previous studies have demonstrated transgenerational male

infertility and altered sperm motility is observed in the vinclozolin

F3 generation males [1,29]. Alterations in sperm epigenomes have

also been linked to male infertility and other disease [63,64,65].

Results of the current study indicate the molecular basis of this

transgenerational disease and male infertility is directly linked to

the altered epigenetic programming of the PGC and subsequent

germ cells investigated.

A comparison between the germ cell DMR and the differen-

tially expressed genes indicated no significant overlap. This

suggests minimal direct promoter regulation through a DMR for

an adjacent gene at these stages of development. In contrast, some

potential indirect gene associations were identified for both the

E13 and E16 developmental stages between the DMR and

differentially expressed genes, Figure 6. Although negligible direct

promoter associations were observed, previously we have identi-

fied an epigenetic control region that may allow a DMR to distally

regulate gene expression over a significant distance [34]. An

‘‘epigenetic control region’’ containing a DMR in proximity to a

long non-coding RNA (lncRNA) may regulate gene expression for

over a 2–5 megabase region [34]. This epigenetic control of gene

expression provides an alternate mechanism for the DMR

identified to regulate distally the PGC and prospermatogonia

Figure 6. Known relationships between genes having DMR intheir promoter regions (grey nodes) and differentially ex-pressed genes (red nodes) in control- compared to vinclozolinlineage F3 generation germ cells from. (A) E13 and (B) E16. Genenode shape code: oval and circle – protein; diamond – ligand; circle/oval on tripod platform – transcription factor; ice cream cone –receptor. Grey connecters represent general regulation, blue –expression regulation, purple – binding, green – promoter binding.Network was derived using Pathway StudioTM software.doi:10.1371/journal.pone.0066318.g006



differentially expressed genes observed. Future studies will need to

correlate the cascade of epigenetic and transcriptional events in

the developing germ cells to these types of epigenetic control

regions.

The combined observations demonstrate ancestral exposure of a

gestating female during fetal gonadal sex determination can

promote transgenerational alterations of normal germline epige-

netic and transcriptional programming that leads to the epigenetic

transgenerational inheritance of disease and phenotypic variation.

Observations support a role for disrupted germline epigenetic

programming in the etiology of the epigenetic transgenerational

inheritance phenomenon. Results suggest a cascade of epigenetic

and transcriptional events during germ cell development is needed

to obtain the mature germline epigenome involved in transgenera-

tional transmission of the epigenetic inheritance.

Methods

Animals and ExposuresHsd Sprague DawleyHTMSDHTM female and male rats of an

outbred strain (Harlan) were maintained in ventilated (up to 50 air

exchanges/hour) isolator cages (with dimensions of 10 L’’ W x 19

J‘‘ D x 10 L’’ H, 143 square inch floor space, fitted in Micro-

vent 36-cage rat racks; Allentown Inc., Allentown, NJ) containing

Aspen Sani chips (pinewood shavings from Harlan) as bedding, on

a 14 h light/10 h dark regimen, at a temperature of 70 F and

humidity of 25% to 35%. Rats were fed ad libitum with standard

rat diet (8640 Teklad 22/5 Rodent Diet; Harlan) and ad libitum tap

water for drinking. At pro-estrus as determined by daily vaginal

smears, the female rats (90 days of age) were pair-mated with male

rats (120 days). On the next day, the pairs were separated and

vaginal smears were examined microscopically. If sperm were

detected (day 0) the rats were tentatively considered pregnant.

Monitoring of vaginal smears was continued for diestrus status in

these rats until day 7. Pregnant rats for the treatment group were

given daily intraperitoneal injections of vinclozolin (100 mg/kg

BW/d; Chem Service, West Chester, PA) and an equal volume of

sesame oil (Sigma) on days E8 through E14 of gestation;

Vinclozolin was dissolved in DMSO (Sigma). Pregnant rats for

the control group were given daily intraperitoneal injections of

DMSO (100 ul/kg BW/d) and an equal volume of sesame oil

(Sigma) on days E8 through E14 of gestation [66]. The pregnant

female rats treated with vinclozolin were designated as the F0

generation. All experimental protocols for the procedures with rats

were pre-approved by the Washington State University Institu-

tional Animal Care and Use Committee (IACUC approval #02568-030).

Breeding F1, F2, and F3 GenerationsThe offspring of the F0 generation were the F1 generation. The

F1 generation offspring were bred to other F1 animals of the same

treatment group to generate an F2 generation, and then F2

generation animals were bred similarly to generate the F3

generation animals. No sibling or cousin breeding was performed

so as to avoid inbreeding. Note that only the original F0

generation pregnant females were injected with vinclozolin or

vehicle.

Fetal Gonadal Germ Cell PreparationHarlan Sprague-Dawley rats (Harlan Inc, Indianapolis IN) were

used for all experiments. The rats were kept in a temperature

controlled environment and given food and water ad libitum.

Estrous cycles of female rats were monitored by cellular

morphology from vaginal smears. Rats in early estrus were paired

with males overnight and mating confirmed by sperm-positive

smears, denoted day 0 of pregnancy. Pregnant rats were

euthanized at embryonic day 13 (E13) or 16 (E16) of gestation,

and fetal gonads were collected for germ cell preparations. At E13,

sex was determined by PCR on genomic DNA isolated from

embryo tails using primers specific for the Sry gene as previously

described [43]. At E16, sex was determined on the basis of gonadal

morphology. Germ cells were isolated exclusively from males.

Purified populations of male PGCs (at E13) or type T1

prospermatogonia (at E16) were prepared using a mini StaPut

gradient method as previously described [67,68]. Briefly, fetal

testes were pooled and dissociated by incubation in 0.25% trypsin-

EDTA (Sigma) with vigorous pipetting using a 1000 microliter

pipette tip, and the resulting cell solution was filtered through

100 micron nylon mesh to yield a single cell suspension. This cell

suspension was then loaded onto a 50 ml 2–4% bovine serum

albumen (BSA) gradient prepared in KREBS buffer, and the cells

were allowed to sediment at unit gravity at 4uC for two hours as

described [67,68]. The gradient was then fractionated and aliquots

of the fractions were examined under phase optics to identify those

enriched for the appropriate PGC or prospermatogonial cell types

on the basis of morphological characteristics. The enriched

fractions were pooled to yield the final sample which was $85%

pure for the desired male germ cell type in each case.

RNA Extraction and Microarray Transcriptome AnalysisMessenger RNA was isolated using the TrizolTM (Invitrogen)

method per the manufacturer’s protocol. Messenger RNA was

independently extracted from 3 pools of germ cells (i.e. 3 biological

replicates) per treatment. The mRNA processing and hybridiza-

tions were performed at the Genomics Core Laboratory, Center

for Reproductive Biology, Washington State University, Pullman,

WA using standard Affymetrix reagents and protocols. Briefly,

mRNA was reverse transcribed into cDNA with random primers,

then cRNA was transcribed from the cDNA, and from that, single-

stranded sense DNA was synthesized which was fragmented and

labeled with biotin. Biotin-labeled, fragmented ssDNA was then

hybridized to the Rat Gene 1.0 ST microarrays containing more

than 27,000 transcripts (Affymetrix, Santa Clara, CA, USA).

Hybridized chips were scanned on an Affymetrix Scanner 3000.

CEL files containing raw data were then pre-processed and

analyzed with Partek Genomic Suite 6.5 beta software (Partek

Incorporated, St. Louis, MO) using an RMA and GC-content

adjusted algorithm (Figure S1). The signals from an average of 28

different probes for each transcript were compared to give a single

value. Two-way ANOVA was performed between the germ cell

transcriptomes from F3 generation vinclozolin and control lineage

cells. One factor of variation was treatment and the other was

batch effect. Corrections were made for cell preparation date

batch effect by the Partek software according to the Methods of

Moments [69]. The selection of the differentially expressed genes

was based on the expression change between vinclozolin and

control lineage germ cells limited to p-values ,0.05, expression

fold change .1.2, and the mean difference between vinclozolin

and control un-logged signals .10. CEL files from this study have

been deposited with the NCBI gene expression and hybridization

array data repository (GEO, http://www.ncbi.nlm.nih.gov/geo,

GEO # GSE43559) and can also be accessed through www.

skinner.wsu.edu. For gene annotation, the Affymetrix annotation

file RaGene1_0stv1.na31.rn4.transcript.csv was used unless oth-

erwise specified.



Pathway and Gene Network AnalysisKnown functional relationships among the F3 generation

differentially expressed genes were identified using the KEGG

pathways from the University of Kyoto (Japan) Encyclopedia for

Genes and Genome website (http://www.genome.jp/_eg/) and

Pathway Express (http://vortex.cs.wayne.edu) [63]. Functional

relationships among the F3 generation differentially expressed

genes and genes with changes in DNA methylation were also

interrogated using Pathway Studio software (Ariadne, Genomics

Inc. Rockville MD), using an unbiased, automated survey of

published scientific literature (Global Literature Analysis). This

analysis identifies functional relations among genes, such as direct

binding, up-regulation or down-regulation and also builds sub-

networks of genes and cellular processes based on their inter-

connections.

DNA Extraction and Methylated DNAImmunoprecipitation (MeDIP)

DNA was isolated using the TrizolTM (Invitrogen) method per

the manufacturer’s protocol, from the same germ cell TrizolTM

preparations that were used for RNA isolations. Therefore, three

independent DNA TrizolTM fractions from germ cells per group

were used to obtain three different biological replicates of DNA

samples from each of the two treatment groups. Each of these

DNA samples were then used for methylated DNA immunopre-

cipitation (MeDIP). MeDIP was performed as follows: 1 mg of

genomic DNA was subjected to a series of three 20 pulse

sonications at 20% amplitude. The appropriate fragment size

(200–1000 bp) was verified using 2% agarose gels. The sonicated

genomic DNA was resuspended in 350 ul TE and denaturated for

10 min at 95uC and then immediately placed on ice for 5 min;

100 ul of 5X IP buffer (50 mM Na-phosphate pH7, 700 mM

NaCl, 0.25% Triton X-100) was added to the sonicated and

denatured DNA. An overnight incubation of the DNA was

performed with 5 ug of anti-5-methylCytidine monoclonal

antibody from Diagenode S.A (Denville, NJ) at 4uC on a rotating

platform. Protein A/G beads from Santa Cruz (Santa Cruz, CA)

were prewashed with PBS-BSA 0.1% and resuspended in 40 ul 1X

IP buffer. Beads were then added to the DNA-antibody complex

and incubated 2 h at 4uC on a rotating platform. Beads bound to

DNA-antibody complex were washed 3 times with 1 ml 1X IP

buffer; washes included incubation for 5 min at 4uC on a rotating

platform and then centrifugation at 6000 rpm for 2 min. Beads-

DNA-antibody complexes were then resuspended in 250 ul

digestion buffer (50 mM Tris HCl pH 8, 10 mM EDTA, 0.5%

SDS) and 3.5 ul of proteinase K (20 mg/ml) was added to each

sample and then incubated overnight at 55uC on a rotating

platform. DNA purification was performed first with phenol and

then with chloroform:isoamyl alcohol. Two washes were then

performed with 70% ethanol, 1 M NaCl and glycogen. MeDIP

selected DNA was then resuspended in 30 ul TE buffer. Whole-

genome amplification was then performed with the WGA2 kit

(Sigma-Aldrich) on each MeDIP sample to be used in the

microarray comparative hybridization analysis.

Tilling Array and MeDIP-Chip Bioinformatic and StatisticalAnalyses

Roche Nimblegen’s Rat DNA Methylation 36720K CpG

Island Plus RefSeq Promoter Array was used, which contains three

identical sub-arrays, with 713,670 probes per sub-array, scanning

a total of 15,287 promoters (3,880 bp upstream and 970 bp

downstream from each transcription start site). Probe sizes range

from 50–75 nucleotides in length with a median inter-probe

spacing of 100 bp. Three different comparative (amplified MeDIP

vs. amplified MeDIP) hybridization experiments included in three

sub-arrays were performed by Nimblegen. Each comparative

hybridization experiment contained one biological replicate of a

germ cell whole genome amplified-MeDIP-DNA sample from

each lineage treatment (control or vinclozolin lineages). Vinclozo-

lin lineage MeDIP DNA samples were labeled with Cy3 and

control lineage MeDIP DNA samples were labeled with Cy5. For

each comparative hybridization experiment, raw data from both

the Cy3 and Cy5 channels were imported into R (R Development

Core Team (2010), R: A language for statistical computing, R

Foundation for Statistical Computing, Vienna, Austria. ISBN 3-

900051-07-0, URL http://www.R-project.org), checked for qual-

ity and converted to MA values (M = Cy5-Cy3; A = (Cy5+Cy3)/

2). The following normalization procedure was conducted within

each array. Probes were separated into groups by GC content and

each group was separately normalized between Cy3 and Cy5

using the loess normalization procedure. Normalization curves

were generated specific to each GC group. The arrays were then

normalized across arrays using the A quantile normalization

procedure. Following normalization, each probe within each array

was normalized and M values were replaced with the median

value of all probe normalized M values across all arrays within a

600 bp window. If the number of probes present in the window

was less than 3, no value was assigned to that probe. Each probe’s

A values were likewise normalized using the same procedure.

Following normalization, each probe’s M value represented the

median intensity difference between vinclozolin lineage and

control lineage samples within a 600 bp window. Significance

was assigned to probe differences between vinclozolin lineage and

control lineage samples by calculating the median value of the

intensity differences as compared to a normal distribution scaled to

the experimental mean and standard deviation of the normalized

M. A Z-score and P-value were computed for each probe from

that distribution. The statistical analysis was performed in pairs of

comparative IP hybridizations between vinclozolin lineage (V) and

control lineage I. V1-C1 and V2-C2 gave 715 sites; V1-C1 and

V3-C3 gave 633 sites; V2-C2 and V3-C3 gave 807 sites (multiple

sites exist within a specific DMR). In order to assure the

reproducibility of the candidate DMR obtained, only the

candidate DMR showing significant changes in all three of the

paired comparisons were chosen as having a significant change in

DNA methylation between the vinclozolin lineage and control

lineage samples. This is a very stringent approach to select for

differences, since it only considers those differences found in all

paired analyses.

The DNA sequence motif analysis for the germ cell DMR

identified used the Glam2 tool from MEME SUITE [70] as

previously described [6].

Supporting Information

Figure S1 Sample histograms and box plots for germcell RNA expression microarray probe signal intensityvalues after pre-processing with an RMA, GC-contentadjusted algorithm. Plots for F3 generation control and

vinclozolin lineage germ cells from E13 and E16.

(PDF)

Table S1 Genes differentially expressed in E13 F3generation primordial germ cells. The 25 genes that were

also found among genes differentially expressed in F3 generation

prospermatogonia at E16 are marked by bold font.

(PDF)



Table S2 Genes differentially expressed in E16 F3generation germ cells. The 25 genes that were also found

among genes differentially expressed in F3 generation PGCs at

E13 are marked by bold font.

(PDF)

Acknowledgments

We acknowledge the expert technical assistance of Dr. M. Manikkam and

Ms. R. Tracey in breeding the animals, Dr. M. Savenkova for assistance in

the transcriptome analysis, and Ms. H. Johnson for assistance in

preparation of the manuscript.

Author Contributions

Conceived and designed the experiments: MKS. Performed the experi-

ments: CGB MH EN RB JRM. Analyzed the data: MKS CGB MH EN

RB JRM. Wrote the paper: MKS. Edited the manuscript: MKS CGB MH

EN RB JRM.

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Environmentally Induced Transgenerational …...epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance